A Nonhuman Primate Transplantation Model to Evaluate Hematopoietic Stem Cell Gene Editing Strategies for β-Hemoglobinopathies

Abstract

Reactivation of fetal hemoglobin (HbF) is a promising approach for the treatment of β-hemoglobinopathies and the targeting of genes involved in HbF regulation is under intensive investigation. Here, we established a nonhuman primate (NHP) transplantation model to evaluate hematopoietic stem cell (HSC)-based gene editing strategies aimed at reactivating HbF. We first characterized the transient HbF induction to autologous HSC transplantation in pigtailed macaques, which was comparable in duration and amplitude to that of human patients. After validating function of the HbF repressor BCL11A in NHPs, we transplanted a pigtailed macaque with CD34+ cells electroporated with TALE nuclease mRNA targeting the BCL11A coding sequence. In vivo gene editing levels were low, but some BCL11A deletions were detected as late as 200 days post-transplantation. HbF production, as determined by F-cell staining and γ-globin expression, was slightly increased in this animal as compared to transplant controls. We also provided proof-of-concept results for the selection of edited NHP CD34+ cells in culture following integration of the P140K/MGMT cassette at the BCL11A locus. In summary, the NHP model described here will allow the testing of novel therapeutic approaches for hemoglobinopathies and should facilitate clinical translation.

Disruption of the BCL11A Erythroid Enhancer Reactivates Fetal Hemoglobin in Erythroid Cells of Patients with β-Thalassemia Major

“…In addition, by performing clinical-scale editing, we achieved high editing efficiency for both the coding and the enhancer region in contrast to previously reported results 32 . Furthermore, the high editing levels observed in our bulk cell population are superior to recently reported editing levels in unsorted bulk in vitro cultures with unclear in vivo performance 10 .…”

Therapeutically relevant engraftment of a CRISPR-Cas9–edited HSC-enriched population with HbF reactivation in nonhuman primates

“…These results represent a substantial improvement in engraftment of gene-edited HSPCs as compared to our previous investigations using bulk CD34 + cells treated with zinc finger nucleases (32) or TALENs (15) in the NHP model. Beyond distinct nuclease platforms and the mRNA-based delivery used, these previous studies targeted different genomic loci and used different culture conditions, both of which may also explain the differences in engraftment.…”

“…We first measured the HbF production in all animals by quantifying circulating F cells in PB. In contrast to control transplant recipients that demonstrated a rapid and transient increase in F cells, which returned to a frequency of less than 1.5% at 250 days after treatment (15), all CRISPR-Cas9-edited animals showed an F cell response that stabilized at frequencies of 6 to 18% in PB. This response was sustained for more than 1 year after transplant ( Fig.…”

Aberrant Clonal Hematopoiesis Following Lentiviral Transduction of HSPC in a Rhesus Macaque

“…ZL34’s hemoglobin levels and red blood cell counts remained within the normal range until one year post-transplantation(Figure S1B,E). Analysis of globin chain ratios showed marked upregulation of γ-globin chains(and thus, fetal hemoglobin) in ZL34’s red blood cells long-term, in contrast to the normal dominance of adult β chains by day 100 or later in other transplanted macaques 19 (Figure 1D).…”